It is typical in production of a chemical substance such as a pharmaceutical or biopharmaceutical that the production process involves a series of technically distinct steps or unit operations which occur in chronological order. The goal of such processes is to end up with a target substance purified to a requisite degree for the intended application. This is true for a protein enzyme intended for pharmaceutical application or one intended for use as an industrial catalyst. In many cases the unit operations may include separation or purification steps involving porous or other high surface to volume ratio media, and methods including chromatography or filtration. Such methods are often further defined in regard to target flow-through or target capture—the latter involving a situation where the flow of target substance in solution through a porous media is hindered by its noncovalent chemical interaction with the surface of the porous media. Examples of capture media type interactions include ion-exchange interactions involving charged chemical moieties on the media, hydrophobic interactions involving apolar or other hydrophobic groups, hydrogen bond interactions, van der Waals' interactions including pii-pii overlap interactions between aromatic groups on the media and the target, “mixed mode” interactions where more than one of the above interactions occurs in a controlled manner due to the media possessing ligands offering more than one interactive groups or mixtures of different ligands offering different interactive groups, and affinity interactions. The latter may include boronate-carbohydrate affinities, metal ion affinities for chelating groups, or protein affinities for target substances such as avidin protein for biotin, or protein A for Fc regions of antibodies or other proteins. Affinity interactions typically involve mixed mode interactions with some defined molecular structure serving as the basis for the interaction. As such, affinity interactions are often the strongest noncovalent interaction involved in capture of protein or other biopolymers by porous media such as chromatography or filtration media.
What the above interactions have in common is that they bind target in a manner that localizes it in essentially native hydrated form at the surfaces of media. The media does not have to be porous to effect such capture however porous, capillary bed or other large surface area media allow such interactions to bind a significant amount of target per unit media volume (e.g. milligram per milliliter). It is significant to note that proteins captured at porous media or other surfaces, via the above interactions, often display native enzymatic or other protein activity and so they cannot be considered to have undergone a significant physical change. What the above interactions also have in common is that they are reversible allowing native, hydrated material to be readily eluted (recovered) from the media via an alteration of solution conditions such as pH, conductivity, polarity, temperature, etc. which themselves are nondenaturing of target. It is not uncommon that such capture interactions are used to both purify a target, e.g. by selecting binding or elution conditions which favor target over contaminants. Such contaminants my include undesired forms of the target which differ in size, shape, chemical group structure as a result of incorrect production (including intracellular production) or being altered during the purification process via aggregation, denaturation, oxidation, deamidation or other such phenomena common during processing, storage and formulation (for a recent review see Stability of Protein Pharmaceuticals: An Update, Mark Cornell Manning, Danny K. Chou, Brian M. Murphy, Robert W. Payne and Derrick S. Katayama, Pharmaceutical Research, Vol. 27, No. 4, April 2010, pages 544-575). It is also true that proteins or other targets captured on porous media can be recovered (eluted) at concentrations similar to their concentration on the media. Thus capture is often used to concentrate as well as purify a target.
It is typically desirable, from the perspectives of reducing costs or contamination, to produce substances such as pharmaceuticals, biopharmaceuticals or industrial/diagnostic enzymes in as short a time as possible. However in many instances one may wish to delay process time and consciously insert “hold” or “storage” points into a process. There may be several reasons for doing so. For example in the potential future production of metric ton amounts of antibody biopharmaceuticals it is understood that there may be significant cost advantages to running smaller scale purification strategies on a more-or-less continuous production regime rather than one large scale shorter term campaign (e.g. Very Large Scale Monoclonal Antibody Purification: The Case for Conventional Unit Operations, Brian Kelly, Biotechnol. Prog. 2007, volume 23, pages 995-1008.; see also Review: Future of antibody purification, Duncan Low, Rhona O'Leary, Narahari S. Pujar, Journal of Chromatography B, 2008, volume 848, pages 48-63). Such production methods would supposedly also benefit production of targets for non-pharmaceutical applications such as industrial biocatalysts.
It is also possible that in order to meet such production requirements that standard methods such as chromatography or filtration might be augmented with other methods such as precipitation or crystallization (e.g. Low et al. above; see also Alternatives to Chromatographic Separations, Jorg Thommes and Mark Etzel, Biotechnol. Prog. 2007, volume 23, pages 42-45). However the latter two methods may alter target in an undesired manner (e.g. aggregate formation) and, perhaps more importantly, require significant dilution in specific (e.g. low conductivity) solutions to dissolve the crystals or precipitates and recover the target in hydrated soluble form. In this, such methods present drawbacks similar to the widely used protein storage methods of freeze drying, or freezing in solution. As such they are not as compatible as, for example, adding additional capture chromatography or filtration steps to a standard process already involving chromatography or filtration. This difference is significant as there are many instances where one may wish to hold up or otherwise delay one part of the process of producing a biopharmaceutical or similar target. And such instances are increasing. The example of a large mass antibody fermentation being processed in intermediates was noted above. Another example could be where a process is halted for a relatively short period of time, say overnight, to support lower labor costs by eliminating use of a night shift, or to accommodate an “off line” quality control measurement related to a validation required before further processing. So too, consider the case where purified biopharmaceutical is subjected to different types of polishing in order to facilitate different types of formulation (e.g. fluid versus solid formulation) so that target from the same lot can be used in different applications. Another example of emerging importance is the possibility of a biopharmaceutical or other drug being processed to certain degree of purity at one site and then further processed or formulated at another site. Such sites might be separated by a few tens of meters, a few kilometers, or even a few thousand kilometers. In the latter case the target substance may need to be transported in a format which allows it to be uncoupled and taken “off line” from the process line under sterile conditions, transported long distances under controlled conditions and rapidly reintroduced into the process line.
Today the most common approaches to interim storage of proteins and similar targets involve freeze drying or freezing in solution (see references above, e.g. Manning et al.; and those noted below) which alter the target in terms of its normal active and hydrated state, can effect varying degrees of denaturation, and are often time consuming both in preparation of the target for stabilization and storage and resuspending the target in solution to allow for further processing. Such methods also typically involve adding additives to reduce aggregate formation or chemical alteration of target during storage processing, and which may complicate formulation or even need to be removed later by additional processing prior to formulation.
FIG. 1 provides overview of typical recombinant biopharmaceutical production process and notes major process areas such as clarification, purification, polishing, formulation and delivery. Such an overview applies to a wide range of targets including biopharmaceuticals, plasma derived proteins, industrial enzymes, diagnostic enzymes, etc. The figure also notes three different places where there may be a need to hold up a process while maintaining a protein or other target capable of “capture” in a stable state. It is expected that the need for longer storage stability times may increase the closer one moves from crude preparation to bulk drug substance for formulation. In truth these three examples represent a broad spectrum of needs. What is important to note is if in a process one wishes to stop processing of a protein biopharmaceutical for say four to eight hours it may be difficult to accommodate this need by time consuming operations such as freeze drying or freezing. In the case of freezing it involves taking the protein off line in a solution, adding cryoprotectant additives, freezing down with slow temperature cooling to not denature the protein over five to six hours. When the storage period is over it requires an additional five to six hours to thaw the protein sample and reintroduce it to the process line. Of course when dealing with significant amounts of target freeze-drying and resuspension may require even longer periods of time than freezing and potentially offer even more complex challenges in regard to target alteration or denaturation. In such cases the storage operations, not the desired storage time, negatively impact the potential economic or other benefits of such storage, limiting the ability of the operator to design a flexible and more economic process. This is especially true if additional steps and quality control analyses must be performed to remove, and verify removal of, the stabilizing additives prior to formulation.
One of the most significant challenges faced in production and storage of protein targets is microaggregate formation, in part as such aggregates can induce immune mechanisms that limit efficacy of the biopharmaceuticals. (e.g., Protein Aggregation and Its Inhibition in Biopharmaceutics, W. Wang, Int. J. Pharmaceutics, 2005, volume 289, pages 1-30). Interestingly aggregate formation is still a major challenge even though more is known about formation of such aggregates and their link to other phenomena such as protein denaturation during processing and storage (e. g. above review by Manning et al.). Thus a recent patent filing (Amgen US 2010/0056765) provided various additive mixtures to “inhibit protein aggregate formation induced by physical stresses associated with processing, manufacture, shipping and storing protein solutions, particularly freeze/thaw stress” and noted that “additives to stabilize proteins (during free/thaw operations) suffer from certain disadvantages, for example, the necessity of additional processing steps for additive removal. Further, none of the processes described in the art is suitable for stabilizing proteins during repeated freezing and thawing processes such that no . . . aggregates are formed during the manipulation.” It is important to note that aggregates of identical proteins often occur over broad range of sizes from dimeric protein forms (two proteins self associated) to micron sized aggregates of large numbers of protein molecules but that typically presence of dimers suggests unstable storage or other conditions which will also give rise to larger aggregate forms.
While it is true that polymer based precipitation (flocculation) of proteins appears to maintain them in stable form regarding aggregate formation and such precipitates might be useful in regard to some types of purification storage and formulation (e.g. Millipore US 2009/0232737; Biogen IDEC WO2009/051726, Amgen WO 2009/026122, Genentech US 2008/0193981) such methods still come with the drawbacks noted above.
No storage method is known which is capable of not only holding aggregate levels stable but also reducing aggregate levels.
It is well recognized that modern bioprocess separation media including chromatography media, monolithic media, filtration media often do not denature proteins over the time scales of most separation operations—typically less than 12 hours. The benign effect of such media is recognized even when proteins are covalently localized at the surfaces of such media including agarose based media. (e.g. Glyoxyl agarose: A fully inert and hydrophilic support for immobilization and high stabilization of proteins, C. Mateo et al., Enzyme and Microbial Technology, 2006, volume 39, pages 274-280.). It has recently been noted that proteins can be affinity bound to porous or other media and then stabilized by partial or total drying of the protein bound matrix at temperatures 18 to 42° C. (WO 2009/034204). The reason for stable storage of the unhydrated protein is unknown but may be related to its complexation by polymers on the matrix in manner similar to what occurs in dehydrated polymer-protein precipitates. This may be similar to storage of proteins by drying them sugar solutions such as available in commercially supplied formulations (e.g. READYTOGO™ reagents from GE Healthcare). Use of such technology for large-scale protein production would be hindered by the need to dry (and later rehydrate) the protein in controlled manner and need for specialized equipment.
Therefore, there is a need for an improved method for stable storage of sensitive biological or chemical target substance in hydrated form so that rehydration and solubilization is not required. There is also a need for a method to effectively reduce aggregates for such target substance.